Functional validation of specific targets from the HIS in human breast tumors in vivo. (A) Migratory and average primary tumor cells were isolated in vivo from MDA-MB-231 as well as the patient-derived HT17 and HT39 tumors. Cells were fixed immediately after collection and immunostained for total Smad2/3 complex, with DAPI used as a nuclear counterstain. A representative image for a cell with cytoplasmic Smad2/3 staining from the primary tumor samples and a cell with nuclear accumulation of Smad2/3 from the migratory cell samples is shown. Quantification of total results is shown in the graph, for which the average percentage of cells with nuclear Smad2/3 accumulation over total number of cells (by DAPI count) was calculated for each xenograft. Error bars: SEM, *P < 0.05 (Student t test), n = 10 to 50 cells per sample; samples from at least three different mice. ( B) In vivo invasion and intravasation were measured in mice bearing either orthotopic MDA-MB-231-GFP tumors (MDA231) or patient-derived HT17 and HT19 tumors, shortly after treatment with specific inhibitors or blocking antibodies. In vivo invasion is plotted as average number of migratory cells collected per microneedle. Intravasation is plotted as average number of circulating tumor cells per milliliter of blood. Results are shown for mice that received treatment with either vehicle control or specific inhibitor: neutralizing antibody specific to human IL8, PTPN11 specific inhibitor NSC87877, TGF-β receptor-specific inhibitor SB431542, NPM1-specific inhibitor NSC34884, or MYC-specific inhibitor 10058-F4 (negative control). Bars, average number of cells; error bars: SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (Student t test for each condition relative to its vehicle control); n ≥ 6 microneedles from at least four mice for the in vivo invasion assay; n ≥ 6 mice for the intravasation assay. (C) mRNA expression of MDA-MB-231 cells transfected with siRNA for genes SMAD2, IL8, PTPN11, NPM1. Shown is expression for each target gene by its respective siRNA relative to the nontargeting control siRNA (si-control). Error bars: SEM, *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test); n = 3 separate experiments for each siRNA. (D) In vitro invasion over Matrigel-coated transwells was measured for MDA-MB-231 cells, either transfected with siRNA to the genes indicated or with specific inhibitors or blocking antibodies. Shown is the relative invasion for each condition toward the appropriate control. Error bars: SEM, *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test); n = three separate experiments for each condition with duplicate transwells per experiment.